Functional disruption of macrophage migration inhibitory factor (MIF) suppresses proliferation of human H460 lung cancer cells by caspase-dependent apoptosis

The human non-small cell lung cancer cell-lines H460 and A549, were both obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat Sen University, Peoples Republic of China. H460 cells were cultured in RPMI-1640 medium (GIBCO, USA) and A549 cells were cultured in DMEM medium (GIBCO, USA) where both cultures were supplemented with 10% newborn calf serum. Both cell-lines were maintained in a fully humidified incubator at 37°C and an atmosphere of 5% CO₂ in air.

For transfection experiments, two independent siRNA species were designed to target knockdown of functional MIF expression and were obtained from Invitrogen (San Diego, CA, USA). The sequences of each miRNA species were:

The negative control siRNA was also obtained from Invitrogen, and had the following sequence:

Sense 5^′-GCGCGCUUUGUAGGAUUCGdTdT −3^′, Antisense 5^′ -CGAAUCCUACAAAGCGCGCdTdT −3.

Cell-lines at an exponential phase of proliferation were seeded into 6-well culture plates, 1.5 × 10⁵ cells per well. When the confluence of the cells approximated 30-40% of the available surface area of the culture wells, the cells were transfected with 50 pmol/ml siRNA (a dose that was found to be optimal in dose-dependent experiments), using lipofectamine 2000 reagent (Invitrogen, USA) following the manufacture’s protocol. Cells transfected with the negative control siRNA were used to control for the specificity of the miRNA MIF knockdown studies. The transfection efficiency was monitored by observation of the frequency of immunofluorescent positive cells by microscopic examination.

Approximately 5 × 10³ cells per well were seeded into 96-well plates and transfected with MIF siRNA or negative control siRNA, both at a dose of 50 pmol/ml using lipofectamine 2000 reagent (Invitrogen, USA) following the manufacture’s protocol. At the indicated time points of 24 h, 48 h and 72 h post-transfection, the extent of cellular proliferation was measured by MTT assay. This was done by adding MTT reagent (20 μl) to each well and incubating the plates for an additional 4 h at 37°C. At the conclusion of the assay, the medium was aspirated, and dimethyl sulfoxide (150 μl) was added to each well to dissolve the formazan product following metabolism of the MTT reagent. Absorbance values of the formazan product were measured at a wavelength of 490 nm (A₄₉₀). Each experiment was repeated at least three times.

A plate-cloning assay was also carried out 24 h after the transfection of MIF siRNA, In this assay, H460 cells were collected, trypsinized, and plated into 6-well plates of 200 cells per well. Cells were cultured in complete medium (2 ml/well) continuously for 10 days, following which they were fixed, stained, and observed for the formation of visible cultured cell clones by light microscopy. Aggregation of ≥50 cells was considered as a clone. The percentage of clone formation was calculated according to the following equation:

The H460 cell-line was seeded at a density of 1.5 × 10⁵ cells/well into 6-well culture plates under conditions described above. Cells were treated with either MIF siRNA or NC siRNA (for the control group) for a period of 48 h of continuous culture in the presence of these siRNA species. At the conclusion of the assay, cells were harvested by trypsinization, washed three times in PBS and resuspended in 0.5 ml PBS. Immediately after resuspension of the cells, propidium iodide (PI) and a FITC-conjugated monoclonal antibody specific for Annexin V (KaiGi Technology, Guangzhou, China) were incubated with the cells at 4°C for a period of 30 minutes. Cell apoptosis was measured using Flow cytometry (Becton Dickinson Biosciences, Inc., NJ, USA).

Treated H460 cells were lysed in a lysis buffer supplemented with a protease inhibitor cocktail (Tissue or Cell Total Protein Extraction Kit, Shanghai, China). After 10 min incubation on ice, the cell suspension was centrifuged at 12000 g for 20 min at 4°C. Soluble protein fractions were then analyzed by Western immunoblotting performed as follows: First, protein samples were resolved on 12-15% SDS-PAGE gels. The protein bands were transferred onto PVDF membranes (Millipore, USA) which were then blocked overnight in TBS-Tween 20 (TBST) buffer containing 5% w/v skimmed milk proteins. The membranes were washed three times with TBST for 10 min each. Second, the membranes were incubated with an appropriate dilution of a specific primary antibody targeted against MIF (Abcam, USA), caspases-3, -4 and −8 and AKT (all obtained from Cell Signaling Technology, USA) with gentle shaking overnight at 4°C. Thirdly, the membranes were washed thoroughly with TBST and incubated with a HRP-conjugated secondary antibody (Cell Signaling Technology, USA) for 1 h at room temperature. Finally, after the membranes were washed with TBST, the resolved and transferred protein signals were detected by enhanced chemiluminescence (ECL). The stained bands were scanned and the relative optical densities measured for semi-quantitation of the relative expression levels of each ECL detected protein band.

Prior studies have reported that MIF is over-expressed in human lung adenocarcinomas [12, 13, 29]. We evaluated the expression of MIF in H460 and A549 cells, and found that the expression of MIF was highest in H460 cells (Figure 1). In pilot experiments, we found that a concentration of 50 pmol/ml of specific MIF siRNA was optimal for disrupted expression of MIF (see below). The transfection efficiency was monitored by immunofluorescence observation and we found that the transfection efficiency was more than 70% (Figure 2).

In addition, we found that at siRNA concentrations greater than 50 pmol/ml, the extent of cell death increased dose-dependently with increasing siRNA concentrations. Therefore, we selected a dose of 50 pmol/ml for optimal transfection of H460 cells with siRNA for all the following experiments.

After incubating the cells for 48 h with the MIF siRNA species, we determined the relative expression of MIF knockdown by Western immunoblotting (Figure 3). We found that the expression of MIF protein was significantly reduced in the cells transfected with MIF siRNAs as compared those cells treated with the NC siRNA. These observations suggested that MIF siRNAs could dampen the functional expression of MIF in H460 cells very efficiently (Figure 3).

Using the MTT metabolic and viability assay, the cellular proliferation and viability of treated H460 was determined (Figure 4A). We found that in H460 cells treated with MIF siRNA displayed significantly reduced proliferation as compared with cells treated with the NC siRNA control sequence (p <0.05). In addition, the plate cloning assay indicated that MIF siRNA inhibited the proliferation of H460 cells (Figure 4B) which was concordant with the data obtained by MTT assay.

We also assessed the cell cycle dynamics (Figure 5) of H460 cells treated with the NC siRNA control (A), and following treatment with either MIF siRNA1 (B), or MIF siRNA2 (C). In these cell cycle analyses, we could not determine any significant effect on the major cell cycle compartments wherein the G0/G1 peaks, S-phase and G2/M phases of the cell cycle appeared quite similar (Figure 5). Nonetheless, collectively, these data suggest an important functional role for MIF in lung tumor cell proliferation.

Consistent with the observations indicating apoptotic modes of cell death in H460 cells treated with MIF siRNA (Figure 5), we next pursued multi-parameter flow cytometric analysis of siRNA transfected H460 cells to obtain more sensitive and quantitative details of a possible apoptotic mode of cell death (Figure 6A and B). Following 48 h of culture of MIF siRNA transfected H460 cells, flow cytometry was used to quantify the expression of Annexin-V in the absence of PI staining following MIF siRNA treatment (Figure 6A), a condition which indicated apoptosis. We found that in cells treated with both MIF siRNA1 and MIF siRNA2, displayed significantly higher levels of Annexin-V staining (18.09 ± 0.41% and 23.38 ± 2.67% respectively) as compared their negative control siRNA treated counterparts (5.87 ± 1.05%, p < 0.05, Figure 6A and 6B). These observations collectively suggested that MIF may serve important regulatory roles in apoptosis.

In an attempt to elucidate the putative mechanisms responsible for the possible anti-tumor effect of MIF siRNA, we assessed the relative expression levels of proteins thought to be associated with apoptosis and cellular proliferation. We found that the expression of the cleaved band (i.e. caspase-3 and −4) of pro-caspase-3 and −4 were significantly higher in the MIF siRNA groups (Figure 7A), while the expression of both caspase-8 (Figure 7A) and Akt in H460 cells remained unaltered (Figure 7B) irrespective of the siRNA treatment. Collectively, these observations suggest that MIF siRNA not only blocks cell proliferation of H460 cells, but also promotes an apoptotic mode of programmed cell death that may be dependent, at least in part, on enhanced cleavage of pro-caspases-3 and −4 to their mature functional procaspase counterparts (i.e. caspase-3 and −4) (Figure 7A).